U.S. patent number 6,777,198 [Application Number 09/972,882] was granted by the patent office on 2004-08-17 for assay method and kit therefor.
This patent grant is currently assigned to Pharmacia Diagnostics AB. Invention is credited to Ib Mendel-Hartvig, Lena Odelstad.
United States Patent |
6,777,198 |
Mendel-Hartvig , et
al. |
August 17, 2004 |
Assay method and kit therefor
Abstract
The invention relates to a method of determining an analyte in a
sample, especially a high concentration analyte, comprises the
steps of: a) contacting the sample with a specified amount of a
receptor which binds specifically to the analyte to form an
analyte/receptor complex, said specified amount of receptor being
in excess of that required to bind all analyte in the sample, b)
isolating on a solid phase a specified fraction of the amount of
receptor contacted with the analyte, including analyte/receptor
complex and unreacted receptor, c) detecting the amount of
analyte/receptor complex in said isolated specified fraction, and
d) from the detected amount analyte/receptor complex, determining
the concentration of analyte in the sample. The invention also
relates to test kits for carrying out the method.
Inventors: |
Mendel-Hartvig; Ib (Uppsala,
SE), Odelstad; Lena (Uppsala, SE) |
Assignee: |
Pharmacia Diagnostics AB
(Uppsala, SE)
|
Family
ID: |
27354612 |
Appl.
No.: |
09/972,882 |
Filed: |
October 10, 2001 |
Foreign Application Priority Data
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Oct 11, 2000 [SE] |
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0003662 |
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Current U.S.
Class: |
435/7.94;
435/7.1; 435/7.92; 435/971; 436/518; 436/524 |
Current CPC
Class: |
G01N
33/538 (20130101); G01N 33/541 (20130101); G01N
33/566 (20130101); Y10S 435/971 (20130101) |
Current International
Class: |
G01N
33/566 (20060101); G01N 33/538 (20060101); G01N
33/541 (20060101); G01N 33/536 (20060101); G01N
033/53 (); G01N 033/543 () |
Field of
Search: |
;435/7.1,7.92,7.94,7.5,971,800,805 ;436/518,501,524,532 |
References Cited
[Referenced By]
U.S. Patent Documents
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4373932 |
February 1983 |
Gribnau et al. |
4623461 |
November 1986 |
Hossom et al. |
4693969 |
September 1987 |
Saxena et al. |
5063081 |
November 1991 |
Cozzette et al. |
5187065 |
February 1993 |
Schutzer |
5296347 |
March 1994 |
LaMotte, III, et al. |
5420016 |
May 1995 |
Boguslaski et al. |
5569608 |
October 1996 |
Sommer et al. |
5573909 |
November 1996 |
Singer et al. |
6183972 |
February 2001 |
Kuo et al. |
6184042 |
February 2001 |
Neumann et al. |
6316205 |
November 2001 |
Guan et al. |
6319676 |
November 2001 |
Nazareth et al. |
|
Foreign Patent Documents
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0 378 391 |
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Jul 1990 |
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EP |
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0 617 287 |
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Sep 1994 |
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EP |
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0105714 |
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Apr 1984 |
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GB |
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58211659 |
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Sep 1983 |
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JP |
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611498863 |
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Jul 1986 |
|
JP |
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98/18968 |
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May 1998 |
|
WO |
|
Other References
Bayer et al (The Avidin-Biotin System, Immunoassay, Chapter 11, pp.
237-267, edited by Eleftherious P. Diamandis, 1996).* .
Maggio et al (Enzyme-Immunoassay, p. 186-187, 1987).* .
Seiichi Hashida et al., Biotechnology Annual Review, vol. 1, pp.
403-451. .
John E. Butler, Journal of Immunoassay, vol. 21(2&3), pp.
165-209, (2000). .
Eiji Ishikawa et al., Molecular and Cellular Probes, vol. 5, pp.
81-95, (1991). .
John E. Butler, Methods, vol. 22, pp. 4-23, (2000). .
Gert Doekes, Occupational and Environmental Medicine, vol. 53, pp.
63-70, (1996)..
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Primary Examiner: Le; Long
Assistant Examiner: Counts; Gary W.
Attorney, Agent or Firm: Dinsmore & Shohl LLP
Parent Case Text
This application claims benefit of provisional application serial
No. 60/241,366 Oct. 19, 2000.
Claims
What is claimed is:
1. A method of determining the presence of an analyte in a sample
comprising the steps of: a) contacting the sample with a known
amount of a receptor which binds specifically to the analyte to
form an analyte/receptor complex, wherein the known amount of
receptor is in excess of an amount of the receptor required to bind
all analyte in the sample, b) isolating on a solid phase a fraction
of the receptor contacted with the analyte, the resulting isolated
fraction of receptor contacted with analyte including the
analyte/receptor complex and unreacted receptor, and the ratio
between the receptor in said isolated fraction and the known amount
of receptor contacted with the sample being in a range of from
about 1:2 to about 1:1000, c) labeling the analyte/receptor complex
in said isolated fraction and detecting the amount of labeled
analyte/receptor complex in said isolated fraction; and d) from the
detected amount of labeled analyte/receptor complex, determining
the presence of analyte in the sample.
2. The method according to claim 1 in which the sample has a
concentration of greater than 1 nmole/litre.
3. The method according to claim 1 or claim 2 in which the sample
is undiluted.
4. The method according to claim 1 or 2, wherein isolating said
fraction of receptor contacted with the sample on the solid phase
comprises providing a solid phase having binding sites incorporated
thereon for the receptor, and after contacting the sample, or an
aliquot thereof, with a liquid phase containing the receptor,
binding said fraction of receptor to the solid phase.
5. The method according to claim 4, wherein all of the receptor
contacted with the sample has reactivity towards said binding sites
on the solid phase, and receptor-binding capacity of the solid
phase is less than solid-phase binding capacity of receptor
contacted with the sample.
6. The method according to claim 4, wherein only the ratio between
the total binding capacity of receptor and binding capacity of
receptor towards said binding sites on the solid phase is in the
range of from about 2:1 to 1000:1.
7. The method according to claim 1 or 2, wherein isolating said
fraction of receptor on the solid phase comprises contacting the
resulting receptor contacted with analyte with the solid phase.
8. The method according to claim 1, wherein the receptor comprises
a first part that binds specifically to the analyte, and a second
part that binds to the solid phase.
9. The method according to claim 8, wherein the solid phase binding
part of the receptor comprises one member of a specific binding
pair, and the other member of the binding pair is immobilized to
the solid phase.
10. The method according to claim 1, wherein in step c) the
analyte/receptor complex is detected by a labeled detection reagent
which binds specifically to the analyte.
11. The method according to claim 1, wherein said solid phase
binding sites for the receptor are immobilized in a reaction zone
of a flow matrix.
12. The method according to claim 1, wherein the receptor is an
antibody or immunoreactive fragment thereof.
13. The method according to claim 10, wherein the detection reagent
is an antibody or immunoreactive fragment thereof.
14. The method according to claim 10, wherein the detection reagent
is labelled by a fluorophore or chromophore.
15. The method according to claim 9, wherein the specific binding
pair is biotin-avidin or biotin-strepavidin.
16. The method according to claim 1, wherein the sample is an
undiluted serum sample.
17. The method according to claim 1, wherein the sample is an
undiluted whole blood sample.
18. The method according to claim 9, wherein the ratio between
receptor in said isolated fraction and the known amount of receptor
contacted with the sample is in a range of from about 1:5 to
1:100.
19. The method according to claim 9, wherein the ratio between
receptor in said isolated fraction and the known amount of receptor
contacted with the sample is no more than about 1:20.
20. The method according to claim 11, wherein said flow matrix is a
lateral flow matrix.
21. The method according to claim 20, wherein said lateral flow
matrix is a membrane strip.
Description
FIELD OF THE INVENTION
The present invention relates to a method of quantitatively or
semi-quantitatively determining an analyte in a sample, especially
a high concentration analyte.
BACKGROUND OF THE INVENTION
For qualitative and quantitative determination of an analyte in a
sample, a so-called sandwich assay is often used, wherein two
receptors directed against different epitopes of the analyte are
incubated with a sample containing the analyte, one of the
receptors being detectable, e.g. through a label conjugated
thereto. In a heterogeneous assay format, the second receptor is
immobilized (e.g. coupled) to a solid phase or provided with a
binder component, such as biotin, capable of binding to the solid
phase, such as an avidin- or streptavidin-coated solid phase.
Especially in case the analyte is present in the sample in a high
concentration, it is customary to dilute the sample before
performing the assay to avoid the use of large and often costly
amounts of immobilized receptor and labelled receptor,
respectively, or to avoid technical difficulties where large
amounts of receptors cannot be used. Such dilution is not only
laborious but also introduces an additional source of error into
the assay.
There is therefore a need of an assay procedure that avoids the
necessity of dilution.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a method of
performing a heterogeneous sandwich assay which permits the
determination of even a high concentration analyte in a sample
without the need to dilute the sample.
It is another object of the invention to provide a method of
performing a heterogeneous sandwich assay which reduces the amounts
of capturing and detection reagents used.
It is still another object of the invention to provide test kits
for carrying out the method.
In one aspect of the present invention there is therefore provided
a method of determining an analyte in a sample, especially a high
concentration analyte found in concentrations>1 nmole/liter,
comprising the steps of: a) contacting the sample with a specified
amount of a receptor which binds specifically to the analyte to
form an analyte/receptor complex, said specified amount of receptor
being in excess of that required to bind all analyte in the sample,
b) isolating on a solid phase, preferably a matrix such as a
membrane strip, a specified fraction of the amount of receptor
contacted with the analyte, including analyte/receptor complex and
unreacted receptor, c) detecting the amount of analyte/receptor
complex in said isolated specified fraction, and d) from the
detected amount analyte/receptor complex, determining the
concentration of analyte in the sample.
In another aspect of the present invention there is provided a test
kit for determining an analyte in a sample, comprising (i) a
specified amount of a receptor substance having a first part which
binds specifically to the analyte, and (ii) a solid phase member
having immobilized thereon a ligand which binds specifically to a
second part of the receptor, the amount of said ligand on the solid
phase member being less than said specified amount of the receptor
substance.
In still another aspect of the present invention there is provided
a test kit for determining an analyte in a sample, comprising (i) a
specified amount of a receptor substance having a first part which
binds specifically to the analyte, only a specified fraction of the
amount of receptor substance having a second part capable of
binding to a specific ligand, and (ii) a solid phase member having
said specific ligand immobilized thereon.
In yet another aspect of the present invention there is provided a
test kit for determining an analyte in a sample, comprising (i) a
first specified amount of a receptor substance, and (ii) a solid
phase member having immobilized thereon a second specified amount
of the receptor substance.
While it is preferred to use the method and test kit for
quantitative determination of analytes of interest, they may also
be used for semi-quantitative and qualitative determinations.
DETAILED DESCRIPTION OF THE INVENTION
The essence of the present invention resides in binding all analyte
present in a sample to an analyte-specific receptor, isolating a
minor fraction of the analyte-receptor complex formed on a solid
phase, detecting the amount of isolated analyte-receptor complex,
and from this detected amount of analyte on the solid phase
determining the total amount of analyte in the sample. According to
the invention, this may be accomplished in various ways.
In one embodiment of method of the invention, all analyte is bound
by contacting the analyte-containing sample with a solution
containing an excess of a first receptor (R1) which in addition to
affinity to the analyte has affinity to a ligand (L), whereupon a
minor fraction of the analyte-receptor complex is bound to a solid
phase having the ligand (L) immobilized thereto. This binding of
the minor fraction may be achieved by either (i) using a limited
(specified) amount of ligand (L) to extract a fraction of the
analyte-receptor complex (and unreacted receptor), or (ii) by using
a first receptor (R1) only a minor (specified) fraction of which is
capable of binding to the ligand (L) to extract the desired
fraction of the analyte-receptor complex (and unreacted receptor).
In the latter case (ii), the amount of immobilized ligand (L) is
usually in excess of the amount of the first receptor capable of
binding to the ligand (L). The amount of analyte/receptor complex
bound to the solid phase is then detected, usually by contacting
the solid phase with a detecting agent in the form of a labelled
binder for the analyte, such as a labelled second receptor
(R2).
In the first case (i) above, the amount of immobilized ligand (L)
that can bind to the analyte-specific receptor (R1) is a specified
fraction of the amount of analyte-specific receptor (R1) contacted
with the sample, and therefore the ratio of detected analyte on the
solid phase to the total amount of analyte in the sample will
correspond to the ratio of immobilized analyte-binding ligand (L)
to the total amount of added receptor (R1), thereby permitting the
analyte concentration in the sample to be calculated.
In the second case (ii) above, the amount of analyte-specific
receptor (R1) that can bind to immobilized ligand (L) is a
specified fraction of the total amount of receptor (R1), and
therefore the ratio of detected analyte on the solid phase to the
total amount of analyte in the sample will correspond to the ratio
of analyte-specific receptor (R1) capable of reacting with ligand
(L) to the total amount of receptor (R1), thereby permitting the
analyte concentration in the sample to be calculated.
The term "receptor" as used herein refers to active analyte-binding
receptor, and, where relevant, active ligand-binding receptor,
respectively, and is not meant to include such receptor in an
inactive or non-binding state. Likewise, the term receptor-binding
ligand refers to active receptor-binding ligand and is not meant to
include such ligand in an inactive or non-binding state.
The term "amount" as used herein usually means binding capacity.
Thus, for example, when it is stated that the amount of
analyte-specific receptor is in excess of the amount of analyte, it
means that there is more analyte-specific receptor than necessary
to bind all analyte. Usually, there is a 1:1 reaction ratio between
e.g. the analyte and the analyte-specific receptor, or between the
analyte specific receptor and the immobilized receptor-binding
ligand. In such a case, the binding capacities of the respective
species correspond to their molar amounts. Other reaction ratios
are, however, also possible. For example, the immobilized ligand
may be capable of binding more than one analyte-specific
receptor.
In another embodiment of method of the invention, the sample is
contacted with analyte-specific receptor (R1) provided both in
solution and, in a minor fraction, immobilized to a solid phase,
thereby permitting a minor fraction of analyte present in the
sample to be bound to the solid phase. If the ratio of the amount
of receptor (R1) in solution to the amount of immobilized receptor
(R1) is known, the analyte concentration in the sample may be
calculated from the detected amount of analyte bound to the solid
phase.
It is readily seen that the above procedure gives the same effect
as diluting the sample. In addition to the dilution step being
avoided, which, of course, is of advantage to the operator, one
obtains a considerable saving in reagents, i.e. both the reagent
for capturing the analyte on the solid phase and the detecting
agent, the latter often being costly. In this connection, it is
also to be noted that in the assay of the invention, the reaction
between analyte and receptor takes place in solution where almost
all receptors are active rather than at a solid phase surface as in
a corresponding conventional assay where only about 10-20% of
immobilized receptor will react (Butler, J. E., et al, Molecular
Immunology, Vol. 30, No. 13, pp. 1165-1175, 1993).
The required ratio between the total binding capacity of
analyte-specific receptor contacted with the sample and (i) the
binding capacity of receptor-binding ligand that is immobilized to
the solid phase when this is limited, or (ii) the ligand-binding
capacity of the analyte-specific receptor when this is limited, is
readily determined by the skilled person depending inter alia on
the particular analyte to be determined and the particular assay
format used and may be chosen within wide limits. Usually, this
ratio is from about 2:1 to about 1000:1, especially from about 5:1
to about 100:1, preferably more than about 10:1, more preferably
more than about 20:1.
The excess of analyte receptor relative to the amount of analyte in
the sample is also readily determined by the skilled person for
each specific case.
The receptor contacted with the sample is usually of the dual
receptor or bireactive binder type having one part that
specifically binds to the analyte and another part which
specifically binds to the ligand immobilized on the solid phase
surface. The analyte binding part may, for example, be an antibody
(monoclonal or polyclonal) or an active fragment thereof (including
recombinant antibodies and fragments) or nucleic acids whereas the
ligand-binding part may be one member of a specific binding pair.
Exemplary such specific binding pairs include immunological binding
pairs, such as antigen-antibody and hapten-antibody, biotin-avidin
or -streptavidin, lectin-sugar, hormone-hormone receptor, and
nucleic acid duplex. For example, the solid phase may have
streptavidin immobilized thereto, and the receptor for the analyte
may be biotinylated. To avoid immunoprecipitation at high analyte
concentrations, it may be preferable to use monovalent
receptors.
While the analyte preferably is a molecule present in
concentrations>1 nmole/liter in a sample, the analyte may, of
course, be any substance for which there exists a naturally
occurring analyte-specific binding member or for which an
analyte-specific binding member can be prepared.
Analyte that has been captured by the solid phase is usually
detected by reaction with a labelled specific binder for the
analyte. Such a labelled binder may be a conjugate comprising a
detectable label covalently or non-covalently attached to the
specific binding member, "label" referring to any substance which
is capable of producing a signal that is detectable by visual or
instrumental means, particularly a fluorophore or chromophore.
The sample is usually of biological origin, for example blood
(serum, plasma, whole blood), saliva, tear fluid, urine,
cerebrospinal fluid, sweat, etc. The invention is, of course, also
applicable to other types of samples, such as fermentation
solutions, reaction mixtures, etc. Especially, however, the sample
is an undiluted serum or whole blood sample.
While the present invention is generally applicable, it may
advantageously be used in solid phase assays of the
immunochromatograpic type. Such assays use a device comprising a
plate-shaped flow matrix of bibulous material, usually a membrane
strip, such as of cellulose nitrate or glass fiber, in which liquid
can be transported laterally (i.e. in the plane of the strip) by
capillary forces in the membrane. The membrane usually has a sample
application zone, and a detection (or reaction) zone downstream of
the sample application zone. In the detection zone, usually a
capturing reagent for the analyte is immobilized. To conduct an
assay, the application zone is contacted with the liquid sample to
be assayed for the analyte of interest. The device is maintained
under conditions sufficient to allow capillary action of liquid to
transport the analyte of interest, if present in the sample,
through the membrane strip to the detection zone where the analyte
is captured. The capillary liquid flow is usually insured by an
absorbing pad or the like at the downstream end of the strip. A
detection reagent, usually labelled, is then added upstream of the
detection zone and interacts with captured analyte in the detection
zone, and the amount of captured analyte is measured. Often, the
detection reagent is pre-deposited in or on the membrane strip,
e.g. in the form of diffusively movable particles containing
fluorophoric or chromogenic groups, either upstream of the sample
application zone or between the sample application zone and the
detection zone.
In an immunochromatographic assay according to the invention, the
receptor is added to the sample either before applying the sample
to the membrane strip, or may be pre-deposited in or on the
membrane strip upstream of the detection zone.
A test kit for carrying out the method of the invention may
comprise such a membrane having (i) immobilized in or on the
membrane a ligand which binds specifically to the receptor, and
(ii) dissolvably pre-deposited in or on the membrane a specified
amount of analyte-specific receptor. The amount of the ligand on
the solid phase member is less, and usually considerably less than
that required to bind the specified amount of the receptor
substance.
In another embodiment of test kit, only a fraction of the
analyte-specific receptor is capable of binding to the immobilized
ligand. Such a kit may comprise (i) immobilized in or on a membrane
a ligand which binds specifically to the receptor, and (ii)
dissolvably pre-deposited in or on the membrane a specified amount
of analyte-specific receptor substance, only a specified fraction
of which is capable of binding to the immobilized ligand.
Still another embodiment of test kit may comprise (i) dissolvably
pre-deposited in or on a membrane a first specified amount of
analyte-specific receptor substance, and (ii) immobilized in or on
the membrane a second specified amount of the analyte-specific
receptor substance.
In an alternative embodiment, the solid phase is a solid phase
well, such as a microtiter plate well. Such of test kit may
comprise a solid support having one or more wells with the second
amount of analyte binding receptor immobilized therein and with the
first amount of analyte-binding receptor dissolvably pre-deposited
in the well or in close contact with the well.
In the following, the invention will be illustrated in more detail
by a specific non-limiting Example.
EXAMPLE 1
Immunoassay for C-reactive Protein (CRP) in Undiluted Serum Samples
Measuring Range 10-200 mg/l
Principle
Sample is mixed with biotinylated anti-CRP-fab in excess and the
mixture is applied to a test strip having a deficient amount of
streptavidin in the reaction zone. After an intermediate wash,
anti-CRP fluorophore-conjugate is added and after a wash, conjugate
that has bound to the reaction zone is measured. Since only a small
part of the biotinylated anti-CRP-fab can bind to the reaction zone
the consumption of the fluorophore conjugate is reduced
considerably.
Test Strips
5.times.48 mm nitrocellulose membranes (Whatman, porosity 8 .mu.m)
on a polyester backing were used. The strips had a sample
application zone at one end and a downstream reaction zone with
immobilized streptavidin in an amount capable of binding
approximately 6% of biotinylated anti-CRP added in the assay
procedure.
Samples
CRP-containing samples of varying CRP concentration were prepared
from a 200 mg/l of recombinant CRP (Fitzgerald) in hCRP depleted
serum.
Procedure
15 .mu.l of biotinylated anti-CRP-fab (monovalent fab-fragment of
monoclonal antibody) and 15 .mu.l of CRP-containing serum were
mixed and the mixture was applied to the application zone of the
membrane strip. The amount of biotinylated anti-CRP-fab was 3 .mu.g
per test strip, which is a 2.times. molar excess of anti-CRP in
relation to the standard 200 mg/l CRP. After an intermediate wash
with 15 .mu.l of test buffer (50 mM borate buffer pH 8.0, 3% BSA,
5% sucrose, 0.15 M NaCl, 0.005% CaCl.sub.2, 0.05% NaN.sub.3), 15
.mu.l of detection conjugate solution [3 .mu.g of anti-CRP
monoclonal antibody (Fitzgerald) coupled to 0.1 .mu.m
TransFluoSpheres-SO.sub.4 /CHO (633/720 nm) (Molecular Probes
Inc.), the above test buffer] were added, followed by wash with
2.times.15 .mu.l of test buffer. The fluorescence of the strip was
then measured. The results are shown in Table 1 below.
TABLE 1 CRP conc. Peak area obtained (mg/l) (V .times. mm) 0 0.08 0
0.07 10 2.56 10 2.50 30 3.62 30 4.01 100 5.24 100 4.87 200 6.28 200
5.82
EXAMPLE 2 (COMPARATIVE)
Immunoassay for CRP in Serum Samples Diluted 1/20 Measurement Range
10-200 mg/ml
Principle
Sample is diluted in test buffer and applied to test strips having
an excess of anti-CRP in the reaction zone. Anti-CRP
fluorophore-conjugate is then added followed by a wash, whereupon
conjugate that has bound to the reaction zone is measured. Sample
dilution is necessary to avoid unreasonably large amounts of
anti-CRP in the reaction zone as well as in the detection
conjugate.
Test Strips
5.times.48 mm nitrocellulose membranes (Whatman, porosity 8 .mu.m)
on a polyester backing were used. The strips had a sample
application zone at one end and a downstream reaction zone with 2.6
.mu.g immobilized anti-CRP monoclonal antibody (Fitzgerald), which
is a 13.times. molar excess in relation to a standard 10 mg/ml CRP
serum.
Samples
CRP-containing samples of varying CRP concentration were prepared
from a 200 mg/l of recombinant CRP (Fitzgerald) in hCRP depleted
serum.
Procedure
15 .mu.l of CRP-containing serum diluted 1/20 in test buffer (50 MM
borate buffer pH 8.0, 3% BSA, 5% sucrose, 0.15 M NaCl, 0.005%
CaCl.sub.2, 0.05% NaN.sub.3) were applied to the application zone
of the membrane strip. Then, 15 .mu.l of detection conjugate
solution [anti-CRP monoclonal antibody (Fitzgerald) coupled to 0.1
.mu.m TransFluoSpheres-SO.sub.4 /CHO (633/720 nm) (Molecular Probes
Inc.), the above test buffer] were added, the amount of anti-CRP
conjugate being 3 .mu.g per test strip which was a 1533 molar
excess in relation to the highest standard value. The conjugate
addition was followed by a wash with 15 .mu.l The fluorescence of
the strip was then measured. The results are shown in Table 2
below.
TABLE 2 CRP conc. Peak area obtained (mg/l) (V .times. mm) 0 0.41 0
0.60 10 7.51 10 7.130 20 8.86 20 9.42 40 11.97 40 10.67 80 11.70 80
12.91 200 14.27 200 14.16
The above Examples 1 and 2 thus demonstrate that it is possible to
run an assay on undiluted high concentration samples without using
huge amounts of reagents when using the methodology of the present
invention.
* * * * *